The B-raf gene was isolated from a human genomic library and its promoter region characterized. Moreover, a series of cDNA clones were isolated and sequenced. One of these contains the complete sequence of a 72/74 kiloDalton (kDa) B-raf kinase. There is a second form of the kinase, a 95 kDa phosphoprotein, that is currently being characterized. The rat pheochromocytoma cell line (PCI2) expresses several growth and differentiation receptors, including those for epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF), and is extensively used as an in vitro model system to study the mechanisms of growth factor-induced differentiation and proliferation. Depending upon which receptor is activated, either proliferation or differentiation occurs. We have previously shown that a variety of ligands for transmembrane receptor tyrosine kinases regulate Raf-1 activity, including platelet-derived growth factor, colony stimulating factor, insulin, FGF, and EGF. PCI2 cells express Raf-I as well as B - Raf. We wanted to examine whether Raf kinase was involved in this signaling, and whether there was Raf isotype preference in the coupling to a proliferation versus differentiation receptor. Recent experiments demonstrate that stimulation of PCI2 cells with EGF, FGF, or NGF activate B-Raf and lead to a shift in its electrophoretic mobility. This shift is caused by increased phosphate incorporation into the B-Raf protein on serine residue(s). B-Raf phosphorylation occurs within 1.5 minutes of NGF treatment and reaches a maximum level after 10 minutes. These results suggest that B-Raf may play a role in NGFmediated signal transduction. In order to investigate the function of B-Raf in differentiation or proliferation signaling pathways, B-raf stimulation and inhibition experiments will be performed in PCI2 cells employing (1) B-Raf-specific monoclonal antibodies, (2) expression of antisense RNA, and (3) dominant negative, as well as constitutively-activated, B-raf mutants.